Human factor XII is a protein present in blood as a single polypeptide chain in zymogen form. Its concentration in plasma is approximately 29 micrograms/ml. Factor XII is an 80 kDa glycoprotein of beta-globulin mobility composed of 596 amino acids and 16.8% carbohydrate. McMullen et al., J. Biol. Chem. 260:5378 (1985); Fujikawa et al., J. Biol. Chem. 258:10924 (1983). Factor XII participates in the earliest phase of the contact activation system, and is probably the first factor activated during contact activation. It is implicated in mechanisms of inflammation, complement activation, and fibrinolysis. Colman et al., J. Clin. Invest. 73:1249 (1984); Goldsmith et al., J. Clin. Invest. 62:54 (1978).
Human factor XII has been described in the literature as, and is synonymous with, "Hageman factor" and "HF". In vitro activation of factor XII by its physiological activator, kallikrein, results in a cleaved form of factor XII, known as factor XIIa. The latter is composed of two chains, a 50 kDa amino-terminal chain and 28 kDa carboxy-terminal light chain, bonded by disulfide bonds. The factor XIIa light chain contains the enzyme active site or "catalytic triad" of the amino acids serine, aspartic acid and histidine. The factor XIIa heavy chain contains the surface binding domain. Human factor XIIa has been described in the literature as, and is synonymous with, "activated Hageman factor", "alpha-factor XIIa", "HFa" and "factor XIIa HMW".
Factor XII fragment (XIIf), a 32 kDa further cleavage product of factor XIIa, is composed of the 28 kDa light chain bonded through disulfide bridges to a 2-4 kDa carboxyl-terminal fragment of the heavy chain. Factor XIIf has been described in the literature as, and is synonymous with, "factor XII fragment", "beta-factor XIIa", "HFf" and "factor XIIa LMW".
Other cleaved forms of factor XII, produced by kallikrein, trypsin, plasmin, and autoactivation have been described in the literature. Dunn et al., J. Biol. Chem. 257:1779 (1982); Margolis, J. Physiol. (Lond.) 144:1 (1958); Kaplan et al., J. Exp. Med. 133:696 (1971).
In vitro activation of factor XII to the active enzyme factor XIIa occurs on negatively charged surfaces by autoactivation, by proteolytic cleavage, by conformational change, or by some combination of these mechanisms. Non-physiological substances with negatively charged factor XII-activating surfaces include glass, kaolin, celite, dextran sulfate, ellagic acid, sulfatides and cholesterol sulfates. Biological substances that activate factor XII include chondroitin sulfate, heparin and some mast cell proteoglycans. Hojima et al., Blood 63:1453 (1984).
Activation of the contact system through activation of factor XII leads to coagulation, and the release of bradykinin. According to one reaction, surface-bound factor XIIa forms an enzyme-substrate complex with factor XI and catalyzes the conversion of factor XI to its active form, factor XIa. Factor XI is a 160 kDa proenzyme consisting of two identical disulfide-linked polypeptide chains. Factor XI is associated in plasma with the 120 kDa protein high molecular weight kininogen ("HMWK") in a stoichiometric noncovalent complex. HMWK serves as a nonenzymatic cofactor in the factor XIIa-catalyzed conversion of factor XI to factor XIa. Factor XIa in turn activates factor IX, which leads to the sequential activation of the remainder of the coagulation cascade.
According to another reaction, surface-bound factor XIIa cleaves and activates prekallikrein to the active enzyme kallikrein. Prekallikrein is an 88 kDa proenzyme complexed with HMWK. Kallikrein cleaves HMWK to liberate bradykinin, the most potent vasodepressor peptide known. Kallikrein may also cleave factor XIIa once again, forming factor XIIf, which can diffuse back into solution. Kallikrein is also able to dissociate from the activator surface and diffuse into solution to activate more factor XII elsewhere on the surface. Colman et al., J. Clin. Invest. 73:1249 (1984).
While both factor XIIa and factor XIIf activate factor XI, factor XIIa is much more effective, presumably due to some structure on the factor XIIa heavy chain. However, both factor XIIa and factor XIIf are equally . potent in the conversion of prekallikrein to kallikrein.
Gram negative septicemia is a major cause of death and disability among hospitalized patients, with an incidence of several hundred thousand individuals effected each year. Once hypotension occurs in association with circulating bacteria or bacterial endotoxin, the mortality ranges from 40-60%. Another frequent complication is hemorrhage due to disseminated intravascular coagulation. Colman et al., Annu. Rev. Med. 30:359 (1979). The microvascular thrombosis which characterizes this condition leads to consumption of coagulation proteins and platelets, and bleeding due to inadequate hemostasis. Id.
Both the hypotension and the hemorrhage experienced in gram negative septicemia patients is due to activation of the contact activation system by bacteria or its endotoxins. The endotoxins causes activation of factor XII. The active enzyme factor XIIa, in turn acts on its two substrates, factor XI and prekallikrein, resulting in coagulation and bradykinin formation.
Clinical and experimental evidence has indicated that kallikrein activation, and the resulting generation of bradykinin, is responsible for the early phase of hypotension experienced by gram negative septicemia patients. The clinical result of the activation of factor XIIa-induced activation of factor IX, which leads to the sequential activation of the remainder of the coagulation cascade, is disseminated intravascular coagulation with sequela, hemorrhage and/or thrombosis.
At present, the treatment of hypotensive septicemia is difficult due to the danger of volume overload and increased hemorrhage due to heparin treatment of disseminated intravascular coagulation. Since there can be a 50% mortality in this condition in the first 48-hours, new approaches to treatment are needed. What is needed is a specific inhibitor of factor XIIa to control the activation of the contact system and release of bradykinin observed in gram negative septicemia patients.
In a related problem, clinical evidence suggests that side reactions occur in some commercially-prepared blood products due to the presence of an activated factor XII derivative. A fraction of human plasma containing factor VIII prepared by ether fractionation in glass bottles has been observed to give rise to prominent flushing and hypotension in patients, resembling the response to injection of bradykinin, which is a by-product of factor XII activation. Arterial hypotension has been reported to occur in patients given such preparations. Reactions to albumin substitutes have been shown to be due to the presence of factor XIIf in such products. Moreover, the presence of factor XIIf in vivo will cause an immediate 50% drop in blood pressure. Thus, its presence in blood products must be avoided. What is needed is an efficient means for purifying liquids, such as blood products, by removing factor XIIf and related activated factor XII derivatives which may be presented in the liquid.
Kohler and Milstein, Nature 254: 493-497 (1975) were the first to describe the fusion of myeloma cells to immune spleen cells from mice to generate continuous cell lines. These hybrid cell lines, or hybridomas, have characteristics that neither the parental myeloma cells nor parental immune spleen cell possess. Hybridomas are capable of continuously producing homogeneous (monoclonal) antibodies. Prior to the work of Kohler and Milstein, only polyclonal antisera could be obtained.
Although techniques for the production of hybridomas are now extensively described in the literature, e.g., Monoclonal Antibodies, Hybridomas: A New Dimension In Biological Analysis, R. H. Kennet, T. J. McKearn, and K. B. Bechtol, eds., Plenum Press, New York and London (1980), there is no general method for obtaining successful monoclonal antibody-producing hybridomas which can be used with all antigens. Fusion techniques must be varied in each case to obtain hybridomas producing monoclonal antibody to the desired antigen. In order to obtain antibodies specific to a single antigen, laborious purification techniques are required to provide highly purified antigen for immunization. The production of monoclonal antibodies for any given antigen is still a highly empirical process.
Monoclonal antibodies to factor XII directed to an epitope within the heavy chain region of the molecule have been reported. Small et al., Blood 65:202 (1985); Saito et al., Blood 65:1263 (1985); Pixley et al., J. Biol. Chem. 262:10140 (1987). However, because the light chain contains the active enzyme site responsible for catalyzing the cleavage of prekallikrein to kallikrein, and the activation of factor XI to factor XIa, monoclonal antibodies to the heavy chain can at best only partially block these enzyme activities.
Polyclonal antibodies to human factor XII have been reported. Lammle et al., Anal. Biochem. 156:118-125 (1986); Lammle et al., Thromb. Res. 41:747-759 (1986). Such antibodies, being polyclonal, are not specific for a single antigenic determinant.
As used herein, "factor XII light chain" or "light chain region of factor XII" shall mean the 28 kDa carboxy-terminal portion of human factor XII which contains the active enzyme site, without regard to whether said portion is in free form or is covalently linked to other peptide chains, as in factor XII, XIIa and XIIf. Similarly, as used herein, "immunogen containing factor XII light chain" shall include factor XII, factor XIIa, factor XIIf, and other cleaved forms of human factor XII containing the 28 kDa light chain, and shall also include the 28 kDa light chain itself.
As used herein, the expression "monoclonal antibody" (occasionally abbreviated "Mab") shall include not only the intact antibody, but also fragments thereof capable of binding antigen, including, but not necessarily limited to Fab and F(ab').sub.2 fragments.
As used herein, the expression "blood plasma product" means human plasma or fractions of human plasma, such as fractions containing one or more specific proteins, or purified preparations of such plasma proteins.